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This readme file was generated on 2026-05-18 by Rebecca J. Whelan\
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# GENERAL INFORMATION\
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Title of Dataset: Polyclonal antibodies targeting defined epitopes on cancer biomarker MUC16\
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## Author/Principal Investigator Information\
Name: Rebecca J. Whelan\
ORCID: 0000-0002-9293-1528\
Institution: University of Kansas\
Address: Multidisciplinary Research Building, 220G\
Email: rwhelan1@ku.edu\
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# SHARING/ACCESS INFORMATION\
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Licenses/restrictions placed on the data: n/a\
Links to publications that cite or use the data: n/a\
Links to other publicly accessible locations of the data: n/a\
Links/relationships to ancillary data sets: n/a\
Was data derived from another source? No\
Recommended citation for this dataset: Moffitt, et al. Polyclonal antibodies targeting defined epitopes on cancer biomarker MUC16\
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# DATA & FILE OVERVIEW\
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\f1\b ELISA. 
\f0\b0 This folder contains two subfolders (\'93Processed Data\'94 and \'93Raw Data\'94). \'93Raw Data\'94 contains 14 raw data files (.xls) from ELISA experiments. \'93Processed Data\'94 contains corresponding analyzed data (as .xlsx files). \
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\f1\b Flow cytometry. 
\f0\b0 This folder contains two NovoCyte Flow Cytometry Experiment files (as .ncf files) and two corresponding reports (as .pdf). \
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\f1\b SPR. 
\f0\b0 This folder contains two Igor Pro folders (as .pxp files) containing the data used to generate binding isotherms. \
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\f1\b \cf0 Western blots. 
\f0\b0 This folder contains 43 files. Uncropped gel images are included as both .jpg and .tif files. Cropped western blot images are included as pictures in .docx files.\
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# METHODOLOGICAL INFORMATION\
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Selection of MUC16 peptide antigens 
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The tandem repeat sequences of human MUC16 (accession number Q8WXI7) were aligned using ClustalX (28) and PASTA (29). WebLogo (version 2.8.2) was used to visualize conserved and variable amino acids (30). Highly conserved regions (appearing with total or partial fidelity in most or all tandem repeats) were individually examined for their likelihood to be immunogenic. The sequences chosen as peptide antigens, their corresponding numbers and amino acid positions within each tandem repeat, were: CRLTLLRPEKD (Peptide 1, positions 59\'9669), ELGPYTLDRNSLYV (Peptide 2, positions 108\'96121), and EENMQHPGSRKFNT (Peptide 3, positions 21\'9634). 	
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Generation of polyclonal antibodies
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Polyclonal antibodies were produced by Biomatik (Kitchener, ON, Canada). First, three peptide antigens were synthesized by Biomatik with user-defined sequences (Peptide 1: CRLTLLRPEKD, Peptide 2: CELGPYTLDRNSLYV, Peptide 3: CEENMQHPGSRKFNT. Peptide 2 and Peptide 3 were synthesized with an N-terminal cysteine to enable KLH conjugation.) Standard immunization was performed, with two rabbits (A and B) being immunized with each peptide. Each of the six rabbits was given a unique identifier, as follows. Rabbit RB4385 was immunized with peptide 1; the polyclonal pool produced by this rabbit is referred to as \'931A.\'94 Rabbit RB4386 was immunized with peptide 1; the polyclonal pool produced by this rabbit is referred to as \'931B.\'94 Rabbit RB4387 was immunized with peptide 2; the polyclonal pool produced by this rabbit is referred to as \'932A.\'94 Rabbit RB4388 was immunized with peptide 2; the polyclonal pool produced by this rabbit is referred to as \'932B.\'94 Rabbit RB4389 was immunized with peptide 3; the polyclonal pool produced by this rabbit is referred to as \'933A.\'94 Rabbit RB4390 was immunized with peptide 3; the polyclonal pool produced by this rabbit is referred to as \'933B.\'94 Pre-immunization serum (0.5 mL/rabbit) and post-immunization antiserum (~20 mL/rabbit) were collected. Pre-immunization serum was found by the vendor to not bind to the target peptide antigen in ELISA (OD < 0.2 at 1:1000 dilution). Antigen affinity purification was performed on a portion of the post-immunization serum. The resulting purified antibodies were confirmed by the vendor to bind to their target peptide antigen via ELISA (dilution series 1:8000\'961:128,000) and delivered in lyophilized form. Upon receipt, lyophilized proteins were reconstituted in distilled water and stored at 4\uc0\u730 C until use. Before performing affinity assays, the protein concentration in pre-immunization serum, post-immunization serum, and reconstituted affinity-isolated antibody solutions were determined by Qubit fluorescence protein quantification (Thermo, Waltham, MA, USA). Test solutions that were measured in affinity experiments were prepared using an appropriate dilution scheme so that the concentration of pre-immunization serum, post-immunization serum, or purified antibody was consistent across all solutions measured in an experiment. 
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Expression and purification of tandem repeats
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MUC16 tandem repeat sequences were obtained using long-read (Nanopore) sequencing as described previously (18) and were cloned in pET-14b vectors (GenScript, Piscataway, NJ, USA) with BamHI and XhoI enzyme restriction sites. The plasmids were transformed into SHuffle T7 Express E. coli cells (New England Biolabs, Beverly, MA) for expression of the N-terminal 6-His tagged protein to enable purification and immobilization. The E. coli cells were grown to late stationary phase at 30\uc0\u730 C in MagicMediaTM E. coli Expression Medium (Thermo) containing 100 \u956 g/mL ampicillin. Cells were lysed by freeze-thaw cycles in liquid nitrogen followed by sonication in lysis buffer (20 mM sodium phosphate, 10 mM imidazole, 300 mM sodium chloride) containing cOmpleteTM protease inhibitor (Thermo). Recombinant repeat proteins were purified on HisPurTM Ni-NTA Resin (Thermo), followed by size-exclusion chromatography using a Superdex 75 10/300 GL column on an \'c4KTA pureTM 25 chromatography system. Three repeats (R14, R18, R19) were unable to be expressed in soluble form using this approach and were therefore not included in this study. Hereafter, the individually expressed MUC16 tandem repeats are referred to as \'93recombinant repeat proteins\'94 or \'93repeats.\'94 The identity of each repeat was confirmed by mass spectrometry, as reported in our previous publication (20). Figure S1 shows a Coomassie blue-stained gel of the repeats.  Concentrations of the repeats in stock solutions were determined by measurements of absorbance at 280 nm on a Nanodrop spectrophotometer (Thermo). Test solutions were prepared by diluting stock solutions to a consistent concentration appropriate to the experiment. 
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ELISA  
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Indirect ELISA was conducted in Pierce Nickel-Coated Plates by first immobilizing recombinant repeat proteins via their 6-His tag to Ni2+ sites on the plate surface. A saturating amount (886.5 ng, 5x well capacity) of each recombinant repeat was dissolved in PBS before immobilization for 1 hour at room temperature (RT). Wells containing HE4 recombinant protein (Fisher) and wells containing no antigen served as negative controls. HE4 was used as a negative control because it\'97like MUC16\'97is an ovarian cancer biomarker. Therefore, in samples from an ovarian cancer patient, both MUC16 and HE4 may be elevated. All recombinant repeat protein\'96antibody combinations were tested in triplicate. After repeat immobilization, three washes with 200 \'b5L PBS-T (0.05% Tween-20) were done before the addition of 100 \'b5L of affinity-isolated polyclonal antibody (1 \uc0\u61549 g/mL in PBS). The primary antibodies were incubated for 1 hour at RT. Three washes with 200 \'b5L PBS-T were done before the addition of 100 \'b5L goat anti-rabbit HRP secondary antibody (Thermo, 1:20000), followed by incubation for 1 hour at RT. After a final set of three washes with 200 \'b5L PBS-T, chemiluminescent signals were developed with SuperSignal ELISA Femto Substrate (Thermo). A SpectraMax M5 plate reader (Molecular Devices, San Jose, CA, USA) was used for detection of relative chemiluminescence units at 425 nm. Indirect ELISA for the pre-immunization and post-immunization serum samples was conducted in a similar manner, the only differences being that the combinations were done in duplicate and after the addition of the serum samples, the number of washes with 200 \'b5L PBS-T (0.05% Tween-20) was increased from three to five and the duration of each wash was increased from 30 seconds to five minutes. In each case, the total protein concentration added to the ELISA plate as primary antibody was 20 \'b5g/mL. Assuming that 10% of the protein concentration is serum is from antibodies, the serum solutions are prepared such that the antibody concentration is 2 \'b5g/mL.  
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Western blot
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Recombinant repeat proteins were separated by size via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an applied voltage of 150 V for 75 minutes on a 16% acrylamide gel and then transferred to a polyvinylidene difluoride (PVDF) membrane using an applied voltage of 20 V for 90 minutes. After transfer, the membrane was blocked with a solution of 5% non-fat milk in Tris buffered saline containing 0.05% Tween-20 (TBS-T) for 1 hour at RT. The membrane was then incubated overnight at 4\uc0\u730 C with a polyclonal antibody solution (1:500 dilution in 5% non-fat milk TBS-T). After incubation, the membrane was washed with TBS-T for 40 minutes (4 rounds of 10-minute washes). The membrane was then incubated at RT with goat anti-rabbit HRP secondary antibody (Thermo, 1:10,000 dilution in 5% non-fat milk in TBS-T) for 1 hour. The membrane was developed using Pierce ECL Western Blotting Substrate (Thermo) and imaged with a ChemiDoc system (Bio-Rad, Hercules, CA, USA). 
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Isolation of MUC16 
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MUC16 was isolated from conditioned cell media of OVCAR3 cells as previously described (31). Briefly, low passage number OVCAR3 cells were validated by Short Tandem Repeat profiling (ATCC, Manassas, VA, USA) and confirmed to be free of mycoplasma contamination using a PCR kit from Genlantis (San Diego, CA, USA). Cells were cultured under standard conditions of temperature, CO2, and humidity in 175 cm2 tissue culture flasks containing RPMI 1640 media supplemented with 10% FBS and 2 mM L-glutamine (Thermo). Cells were grown until they reached 100% confluency at which point spent media was removed and replaced with fresh media devoid of FBS and phenol red. After five to seven days, conditioned media was collected and centrifuged to pellet cell debris. Supernatant (containing MUC16) was spun through a 100 kDa molecular-weight cut-off filter (MilliporeSigma) to concentrate MUC16. Total protein concentration was determined using a Pierce BCA assay (Thermo). CA125 counts were determined using an R&D Systems human CA125/MUC16 assay kit (Biotechne, Minneapolis, MN, USA). 
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Surface plasmon resonance
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Alto Surface Plasmon Resonance Spectrometer, 16-Channel CMD-Carboxyl (CBX) cartridges with Cartridge Fluid, Low Refractive Index Solution (8% Glycerol), High Refractive Index Solution (32% Glycerol), aliquots of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), aliquots of N-hydroxysuccinimide (NHS), Conditioning Solution (10 mM HCl), Quenching Solution (1 M ethanolamine), and Immobilization Buffer (10 mM sodium acetate at pH 5.0) were purchased from Nicoya Lifesciences (Kitchener, ON, Canada). Regeneration solution (10 mM Glycine-HCl at pH 1.5) and Running Buffer (PBS from GrowCells, Irvine, CA, USA supplemented with 0.1% v/v Tween-20) were prepared in-house. Recombinant CA125 (rCA125) expressed in CHO cells was purchased from R&D Systems (Minneapolis, MN, USA). The protein was reconstituted to a final concentration of 250 \'b5g/mL in PBS. All reagents and samples were supplemented with 0.1% v/v Tween-20. Experimental protocols were created using Direct Kinetics with Single-Cycle Titration assay templates in the Nicosystem software. Sample and sensor temperature was held at 25\uc0\u730 C. For the Activities, default droplet settings were used for Startup, Normalize, Clean, and Build Surface. Default droplet times were used for Kinetic Step, except the analyte association droplets, which were extended from 180 s to 300 s. Peptide 2 polyclonal antibodies from rabbit B were diluted to a concentration of 5 \'b5g/mL in 10 mM sodium acetate at pH 5.0 and immobilized onto the CBX CMD cartridges using EDC/NHS coupling. The concentration of EDC and NHS used was 200 mM. Intact MUC16 from conditioned cell media and rCA125 were introduced as analytes. SPR binding data was downloaded from the Nicosystem software. The signal from each concentration of analyte was averaged and plotted against CA125 concentration using Igor-Pro software (Wavemetrics Inc., Oregon, USA). A 1:1 binding model was used for curve fitting. 
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Flow cytometry: cell line and culture conditions
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Ovarian cancer cell lines OVCAR3 and OVCAR8 were cultured in RPMI 1640 supplemented with 10% FBS and 2 mM L-Glutamine under standard conditions (in a humidified 5% CO2 incubator at 37\uc0\u730 C). After reaching ~90% confluency, cells were detached by incubating with 3 mL 0.25% Trypsin-EDTA solution for 5 minutes. Cell density and viability were determined by counting cells stained with Trypan-blue on a hemacytometer. Cells were then resuspended in staining buffer (PBS with 2% FBS and 2 mM EDTA) at a concentration of approximately 350,000 cells per 50 \u956 L.
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Flow cytometry: binding assays
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Five concentrations of the polyclonal antibodies 2B were prepared in staining buffer: 0 \uc0\u956 g/mL, 10 \u956 g/mL, 20 \u956 g/mL and 50 \u956 g/mL. 50 \u956 L of each antibody solution was added to 50 \u956 L of resuspended cells (around 350,000 cells) and incubated for 30 minutes at RT. Cells were washed twice with 200 \u956 L of  staining buffer followed by incubation with 100 \u956 L of 1:500 dilution of FITC-labelled anti-rabbit secondary antibody (Thermo) for 30 minutes at RT. Cells were washed as described before and resuspended in PBS prior to flow cytometry analysis. An \'93only cells\'94 control was included which was not incubated with either the primary or secondary antibody. Samples were run in duplicate on a NovoCyte Flow Cytometer (Agilent, Santa Clara, CA, USA), and 10,000 events were recorded at a medium flow rate.
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